1. Field
The present disclosure relates to methods and kits for determining the presence of ribonucleic acid (RNA) in a sample.
2. Description of Related Art
The detection and characterization of specific nucleic acid sequences and sequence changes have been utilized to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection, the presence of variants or alleles of mammalian genes associated with disease and cancers, and the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations. Characterization of the RNA species involved in normal biological processes may be important to understanding various little known biological processes.
Detection
The detection and characterization of RNA (e.g., messenger RNA, transfer RNA, ribosomal RNA, small nuclear RNA, and other RNAs) is an important tool in many fields including molecular biology, toxicology, and biochemistry. Messenger RNA (mRNA) is an essential functional constituent of a cell; during the process of gene expression, the functional single strand structure of mRNA is synthesized and serves as an intermediate template for the translation process in protein synthesis. The brief existence of an mRNA molecule begins with transcription of DNA into an RNA molecule, and ultimately ends in degradation. During its life, an mRNA molecule may also be processed, edited, and transported prior to translation. Splicing is the process by which pre-mRNA is modified to remove certain stretches of non-coding sequences called introns; the stretches that remain may include protein-coding sequences and are called exons. Sometimes pre-mRNA messages may be spliced in several different ways, allowing a single transcript to encode multiple proteins.
Detection of messenger RNA (mRNA) is critical in diagnostics because it can provide viral load and gene expression information that DNA detection cannot. These factors often give clues about the progression and prognosis of a disease. The current technologies for mRNA detection present a number of problems including expense and potential for contamination.
Reverse hybrid capture is a novel non-target amplification method for RNA detection that can be used to detect specific gene transcripts from biological samples with a very low risk for contamination. This method uses DNA probes that are hybridized to the RNA targets. The created hybrids are then detected with a hybrid capture antibody system.
The most common methods of mRNA detection include Northern blot, ribonuclease protection assay (RPA), and reverse-transcriptase polymerase chain reaction (RT-PCR). However, each of these techniques, while affording some advantages in sensitivity, requires time and material demands. In addition, some techniques require amplification of the target mRNA since total mRNA represents only about 1% of the total RNA and any particular mRNA is a significantly smaller percentage.
Characterization
Currently, reverse transcriptase-polymerase chain reaction (RT-PCR) is widely used to characterize RNA transcripts. However the method has the following limitations: 1) only a limited number of the specific regions can be co-amplified; 2) mutations or alternative splicing can limit the ability of specific primers to detect the RNA; and 3) it is difficult to characterize the mRNA structure in a continuous mode method.